The present invention relates to an electrospray ionization mass analysis apparatus and system thereof, wherein a sample solution eluting out of a low flow rate chromatograph such as a micro liquid chromatograph is led to an electrospray ion (ESI) source and is ionized therein, and the ions generated in this ion source are fed to a mass spectrometer arranged in a highly vacuum space, where the ions are subjected to mass analysis.
In recent years there has been a remarkable growth in biological researches over diversified fields. Especially, protein, peptide and DNA play an extremely important role in the living body, and have been the objects of study by a great number of research workers. Generally, these organic compounds derived from living organism occur in a very small amount in complicated matrices. There has been a growing demand for extracting a very small amount of these biological organic compounds from the living body and analyzing them using a mass spectrometer directly coupled with liquid chromatograph LC/MS apparatus) with a high degree of sensitivity. The LC/MS apparatus is an apparatus for separating a mixture with a liquid chromatograph (LC) and providing qualitative and quantitative analysis using a mass spectrometer (MS) with a high degree of sensitivity. Electrospray ionization (ESI) is typical ionization means used in the LC/MS. The ESI is ionsization technique used under atmospheric pressure and is known as providing soft and highly sensitive ionization. For this reason, this method has come to be used very often for biological analysis.
To ensure stable and highly sensitive measurement of a very small amount of components using the aforementioned ESI, some parameters must be optimized. One of these parameters is the flow rate that determines the amount of solution to be supplied to the ESI ion source. To achieve highly sensitive measurement, the flow rate of the solution flowing through the ESI capillary tube must be kept within a certain range. In ESI, the optimum flow rate is said to lie in the range from 10 nL/min. (10xe2x88x928 L/min) to several xcexcL/min (10xe2x88x926 L/min). If a solution is fed into the ESI capillary tube at a flow rate higher or lower than this level, the ESI ionization will become unstable and anticipated highly sensitive measurement will not be achieved. U.S. Pat. No. 5,504,329 discloses an art for ESI improvement for providing highly sensitive measurement of a very small amount of components. The art disclosed therein was later called Nanospray technique. After the tip of an extra-fine capillary tube made of glass having an outer diameter of about 0.2 mm and inner diameter of about 0.03 mm has been elongated by a burner or sharpened by etching, the nozzle tip is gold plated. The D.C. voltage of about 1 kV supplied from the high voltage source is applied to the tip of the nozzle. The flow rate of a sample solution from a nanospray device ranges from is several nL/min (several 10xe2x88x929 L/min.) to 10 nL/min (several 10xe2x88x928 L/min.). Measurement for more than one hour was enabled by only the sample sucked into the nanospray spray capillary tube. Accordingly, this nanospray technique has come to be used in combination with extra-low flow rate chromatography in CE (Capillary Electrophoresis); further, it has come to be used for extremely highly sensitive measurement of isolated components. The nanospray technique has enabled ESI measurement in the range of flow rate below 10 nL/min.
In the micro LC field, the flow rate is extremely small, below several xcexcL/min. and a big problem is raised by the dead volume of the LC parts and the pipe connection among the parts thereof. When the dead volume between the micro-column and detector is greater for the flow rate, the sample components separated by the micro-column will be dispersed and mixed among them, with the result that separation and sensitivity will be lost a substantially. Further, the dead volume between the LC pump and micro-column will cause a problem of the delay in gradient elution. This requires the dead volume to be minimized.
Gradient elution is a method for quick elution of the sample component by changing the composition of the eluent with the lapse of time. This gradient elution technique is improves the separation of the sample components. This improves the S/N ratio and reduces the measurement time at the same time. Accordingly, LC is used extensively.
In micro LC, even if the start of gradient is specified and multiple pumps have fed out solvent at a predetermined flow rate, a long time is required before the composition of the eluent is changed in the micro-column. This delay raises a problem. This is called a delay in gradient elution.
Assume that a pump 1 is now feeding out solvent A at 20 xcexcL/min. Also assume that a pump 2 starts to feed out solution B at the rate of 0.2 xcexcL/min. at a predetermined time. A mixer and a pipe regionrranged between pumps 1 and 2 and micro-column. If their volume is 5 xcexcL, the delay of gradient will be 5/0.2=25 min. Namely, gradient is effectively started in the micro-column 25 minutes after the pump 2 started to feed solution B. This makes it difficult to ensure correct separation and analysis by micro LC. In order to improve this delay of gradient elution, it is important to reduce the size of the mixer and dead volume. The dead volume can be decreased by reducing the pipe diameter or pipe length. However, reduction of pipe diameter raises a new problem of easy clogging of the pipe. Especially when a biological sample is to be analyzed, a biological macromolecule such as sugar and protein present in the sample as well as NaCl and salts will cause clogging of the pipe. Further, separation of protein requires salt having a high concentration of 100 mM or more to be added to the mobile phase in many cases. This salt of high concentration is deposited in the dead volume of the pipe, with the result that the pipe is clogged in the final stage. Accordingly, the frequently used system in the micro LC is a micro LC system where A semimicro or conventional LC pump is used up to gradient solution feeding, and the eluent is split immediately before the inlet. A great volume (1 mL/min. to 0.1 mL/min.) of solvent is used up to the pump, mixer and pipe, so the dead volume among them can be ignored. In other words, the problem of delay in gradient elution has been solved. The split eluent at a very small flow rate (10 to several xcexcL/min.) is led to the micro column through the injector. This method has a disadvantage that the greater part of solvent must be discarded by the splitter, but it solves the aforementioned problem of the delay in gradient resulting from dead volume, and ensures economical configuration of the system. For these merits, this method has come to be used over a wide range.
The Japanese Application Patent Laid-Open No. 06-13015 discloses ions implantation apparatus for evaluating a trouble such as equipment failure, displacement by comparing with the reference value the status value of a particular peak in a mass spectrum. The Japanese Application Patent Laid-Open No. 10-10109 discloses an apparatus for avoiding damage of the optical detector cell resulting from a clogged flow path in a mass analysis apparatus directly coupled with a liquid chromatograph, the aforementioned mass analysis apparatus being designed to ionize and detect the component leaching therefrom.
The micro LC wherein the solvent is split before the micro column can be said as an extension of the general-purpose LC and semimicro LC technology. So since the micro LC is capable of analyzing a trace quantity of sample, it is expected to find a widespread use in the field of biological technologies. According to this method, however, the major portion of solvent is split and discarded as waste, and the amount of solvent flowing into the micro column is no more than one hundredth to one tenth of the solvent supplied to the splitter. So even if the ESI capillary are clogged by salt or protein and the solvent cannot be led to the micro column, solvent only flows to the waste liquid. Since solvent pressure is released to atmospheric pressure by the splitter, pressure is not changed by clogging of the micro column. Thus, clogging of the analysis column or ESI capillary is not detected, with the result that the sample will be continuously fed from the automatic sampler
Since the clogging of the micro column or piping is not detected. No solvent flows in the vicinity of the injector, and washing is not carried out by solvent, so the automatic sampler and injector will be contaminated by the sample. A large amount of data file from which any mass spectrum or chromatogram cannot be acquired will be stored in the memory of a control data processor. What is more crucial is that precious samples will be consumed in vain by clogging of the micro column. Further, it is not clear when the micro column was clogged, with the result that data reliability will be placed under suspicion.
Further, the aforementioned Laid-open Publication does not disclose any means for detecting the clogging of a capillary tube or ESI nozzle caused by deposition of salts due to low flow rate and for suspending measurement, thereby avoiding waste of samples in the micro LC/MS, or any specific device for predicting the possible clogging of the capillary tube.
The object of the present invention is to provide an electrospray ionization mass analysis apparatus and the system thereof that ensure effective data at all times during measurement, by utilizing means for detecting the clogging of a capillary tube or ESI nozzle caused by deposition of salts due to low flow rate and for suspending measurement, thereby avoiding waste of samples in the micro LC/MS, or by predicting the possible clogging of the capillary tube.
In an electrospray ionization mass analysis apparatus directly coupled with a micro LC, the present invention prevents clogging of the micro LC column, piping and ESI capillary, and records the alarm in the data and stops the system whenever any clogging has occurred, thereby ensuring highly reliable direct connection with the micro LC.
The present invention provides an electrospray ionization mass analysis apparatus wherein;
eluate from a chromatograph is introduced into the capillary tube, and
an electrospray ion source arranged for generating ions under atmospheric pressure generates ions, which are led into a mass spectrometer disposed in a vacuum chamber where the mass spectrum is given. This electrospray ionization mass analysis apparatus is characterized in that the current value of the ion of a specific mass is monitored, and, when this ion current value has reduced below a threshold value, a flag is set to indicate an error.
Further, the present invention provides an electrospray ionization mass analysis apparatus wherein eluate from a chromatograph is introduced into the capillary tube, and an electrospray ion source arranged for generating ions under atmospheric pressure generates ions, which are led into a mass spectrometer disposed in a vacuum chamber where the mass spectrum is given. This electrospray ionization mass analysis apparatus is characterized in that the ion current value of a specific mass is monitored more than once for each sample, and an approximate expression is formed from multiple ion current values monitored subsequent to measurement of multiple samples, to predict the number of sample measurements where the ion current value is below the threshold value, whereby a warning is displayed on a CRT.
The ESI operates as follows: Voltage of several kilovolts is applied between a metallic capillary having an inner diameter of about 0.1 mm and a counter electrode arranged at some distance (about several tens of mm) away therefrom. When a sample solution is led to the metallic capillary and a high voltage is applied, the liquid in the capillary is dielectrically polarized at the capillary outlet by a high electric field formed on the tip of a metallic capillary. In the positive ionization mode, positive electric charge is induced on the liquid surface, while in the negative ionization mode, negative electric charge is induced on the liquid surface.
As a result, a conical liquid called Taylor cone is pulled out into the atmosphere from the capillary outlet by electric field. If electric field is stronger than the surface tension at the tip of the Taylor cone, electrically charged extremely fine droplets are released into the atmosphere from the tip of the Taylor cone. In conformity to electric field, the generated charged droplets fly in the atmosphere toward a counter electrode to repeat collision with molecules in the atmosphere. This allows charged droplets to be mechanically broken, and evaporation of solvent from the droplet surface is promoted so that charged droplets are quickly pulverized. In the final stage, ions in charged droplets are released into the atmosphere. The ion flies in the atmosphere toward a counter electrode and is led into a highly vacuum mass spectrometer through a capillary tube or aperture arranged in the counter electrode where it is subjected to mass analysis.
Further, the present invention provides an electrospray ionization mass analysis apparatus wherein eluate from a chromatograph is introduced into the capillary tube, and an electrospray ion source arranged for generating ions under atmospheric pressure generates ions, which are led into a mass spectrometer disposed in a vacuum chamber where the mass spectrum is given. This electrospray ionization mass analysis apparatus is characterized by sequentially comprising:
a step of introducing the aforementioned sample into the injector and micro column of the chromatograph in that order,
a step of separating the sample for each component and ionizing it after feeding into the aforementioned ion source in conformity to the lapse of time,
a step of repeating mass sweeping with the aforementioned mass spectrometer and storing the collected mass spectra into the control data processor,
a step of measuring the current value (Is) of the ion having a specific mass in the sample, and comparing between the measured Is and the threshold value (It),
a step of continuing measurement if Is exceeds It,
a step of completing measurement if the Is is not below the It by the time the aforementioned measurement terminates, and starting measurement of the next sample,
a step of indicating an error through the control data processor if the error has occurred where the Is is reduced below the It due to sudden reduction of the Is, and specifying the action to be taken to correct the error,
a step of giving a command of suspending start of sweeping to the mass sweep power source of the mass spectrometer to suspend the collection of mass spectra,
a step of recording an error in the data and displaying that warning, and
a step of suspending transmission of the signal for starting the next sample measurement to an automatic sampler.
Further, the present invention provides an electrospray ionization mass analysis apparatus similar to the above characterized by sequentially comprising:
a step of introducing the aforementioned sample into the injector and micro column of the chromatograph in that order,
a step of separating the sample for each component and ionizing it after feeding into the aforementioned ion source in conformity to the lapse of time,
a step of repeating mass sweeping with the aforementioned mass spectrometer and storing the collected mass spectra into the control data processor,
a step of measuring the current value (Is) of the ion having a specific mass, and comparing between the measured Is and the threshold value (It),
a step of continuing measurement if Is exceeds It,
a step of completing measurement if the Is is not below the It by the time the aforementioned measurement terminates, and starting measurement of the next sample,
a step of suspending the collection of mass spectra due to abrupt reduction of the Is,
a step of indicating an error without suspending the collection of mass spectra during the measurement of one sample by liquid chromatograph (LS),
a step recording an error of the Is having reduced below the It, and terminating the data file upon completion of the LC measurement,
a step of instructing suspension of starting the measurement of the next sample if an error is displayed, and
a step of instructing the automatic sampler to start the measurement of the next sample if no error is indicated.
Further, the present invention provides an electrospray ionization mass analysis apparatus similar to the above characterized by sequentially comprising:
a step of introducing the aforementioned sample into the injector and micro column of the chromatograph in that order,
a step of separating the sample for each component and ionizing it after feeding into the aforementioned ion source in conformity to the lapse of time,
a step of repeating mass sweeping with the aforementioned mass spectrometer and storing the collected mass spectra into the control data processor,
a step of measuring the current value (Is) of the ion having a specific mass, and comparing between the measured Is and the threshold value (It),
a step of continuing measurement if Is exceeds It,
a step of completing measurement if the Is is not below the It by the time the aforementioned measurement terminates, and starting measurement of the next sample,
a step of measuring the Is at least once for each supply of the sample immediately before the column is brought into equilibrium by the solvent of the mobile phase prior to supply of the sample,
a step of recording and displaying an error when the Is is reduced below the It,
a step of stopping the measurement and suspending the supply of a new sample, and
a step of continuing the measurement if the Is is above the It.
As described above, the present invention monitors the Na+ ion that surely occurs in the ESI. When it has been reduced below the threshold value, measurement is stopped because clogging is assumed to have occurred. Further, the current value of the Na+ ion is collected for each measurement and the time for reduction below the threshold value is estimated from their changes. This is indicated on the CRT or the like. In other words, in the micro LC and ESI, clogging of the ESI nozzle and capillary tube seriously deteriorates the throughput and data reliability. Sample waste can be minimized and reliability of acquired data can be improved by detecting this clogging and stopping measurement and introduction of the sample. Maintainability can be improved by predicting possible clogging.